The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Heme is an important endogenous substance (haem, iron protoporphyrin IX). There is no International Nonproprietary Name (DCI) for heme. The chemical state and stability of heme are influenced by the solvent and other environmental conditions.
The term "heme" is used generically here. Other terms refer to specific chemical states of heme. The Fe of heme may be either in the ferrous (Fe.sup.2+) or ferric state (Fe.sup.3+) and is coordinated with the 4 pyrrole nitrogens. "Hematin" is a ferric form and refers to heme dissolved in alkaline solution (heme hydroxide) or neutral solution. "Hemin" is another ferric form in which a chloride ion is coordinated to iron at the fifth or sixth position, as occurs when heme is dissolved in an aqueous solution containing HCl. Hemin is highly stable in crystalline form but is poorly soluble. When heme chloride is dissolved in alkaline solution the chloride is replaced by hydroxyl to form hematin, which is poorly stable and readily forms poorly characterized degradation products.
Heme is also stable when bound to proteins or amino acids such as arginine or lysine. Heme arginate is a product of heme and L-arginine (1). Arginine increases the aqueous solubility and stability of heme. Once heme arginate is infused into the blood stream the heme arginate dissociates and the heme moiety is quickly bound to the plasma proteins in monomeric form (1). Therefore, the formulation of heme as heme arginate augments the stability of heme for intravenous infusion and does not change its physiological utilization. Thus the discoveries presented hereinafter for heme arginate are valid also for other heme preparations. The benefit of using heme arginate instead of other presently known heme preparations is its higher stability and consequently consistent efficacy and lack of serious adverse effects known to be caused by degradation products of heme.
Lyophilized hematin (Panhematin.RTM., Abbott) was approved by the U.S. Food and Drug Administration in 1983. Heme arginate (Normosang.RTM., Leiras) was developed in Finland and is now marketed in several countries in Europe for treatment of acute porphyric attacks. Previously, hematin was prepared in research laboratories or hospital pharmacies, usually from outdated human blood. A preparation of heme albumin was developed in Germany.
The use of hematin (either Panhematin.RTM. or hematin prepared in research laboratories) is often complicated by adverse effects, including phlebitis at the infusion site and disturbances in hemostasis (2, 3, 4). Heme arginate seldom produces these effects and therefore is better tolerated. The side effects of Panhematin.RTM. are greatly reduced if the drug is reconstituted with human albumin in equimolar amounts (5). However, this adds considerable expense and the potential risk of administration of a protein product originating from human blood.
Heme and its derivatives have also been reported in the patent literature. The Finnish patent publication Fl 68970 discloses a method for the preparation of heme arginate and heme lysinate and the use of said compounds in the treatment of porphyria. U.S. Pat. No. 5,233,034 relates to a process for the purification of hemin and to a novel hemin derivative, i.e. an 1:3-adduct of hemin and DMI and its preparation. Finnish patent application No. 841199 relates to a method for the stabilization of hematin by use of albumin.
EP 337598 discloses the use of various metalloporphyrins including hemin for the treatment of diseases caused by HIVs. WO 92/02242 describes the use of metalloporphyrins including heme and heme arginate for the treatment of retroviral infections such as AIDS.
Mutagenesis and carcinogenesis
Most cancers are initiated by genetic change. Thus cells of a given cancer can often be shown to have a shared abnormality in their DNA sequence. This does not yet prove that genetic change is an essential first step in the causation of cancer. A more solid argument is that most of the agents known to cause cancer cause genetic change and, conversely, agents that cause genetic change cause cancer. This correlation between carcinogenesis (the generation of cancer) and mutagenesis (generation of genetic change) is clear for chemical carcinogens, ionizing radiation, and viruses (6).
Mutagenesis in a hematopoietic cell may cause clonal expansion of immature hematopoietic cells (blasts) and consequently cause preleukemic conditions (like myelodysplastic syndromes) and leukemias (7). Thus the anti-mutagenic action of heme and its ability to decrease the proportion of blasts in the bone marrow of myelodysplastic syndromes are interrelated and may involve similar mechanisms of action.
Myelodysplastic syndromes and methods for their treatment
The myelodysplastic syndromes (MDS) are a rather heterogeneous group of acquired, multipotent stem cell disorders characterized by ineffective and dysplastic hemopoiesis resulting in symptomatic anaemia, leukopenia, and thrombocytopenia. The marrow progenitor cells show an impaired growth which is not attributable to inhibitors in the marrow. During the course of MDS the dysfunction of the marrow progresses as shown by cytogenetic analysis and precursor cell differentiation becomes more and more impaired. The clinical course is variable, ranging from a stable, mildly symptomatic disorder to one that progresses rapidly to overt acute leukaemia. Bleeding and recurrent infections are the most frequent causes of morbidity and mortality.
Myelodysplasia may be primary, without any recognizable responsible factor, or secondary to exposure to ionising radiation, alkylating agents or some organic solvents (7). In the material from the district of Dusseldorf approximately 5% of the patients with MDS had the secondary type of this disease (8).
In 1982 the FAB group proposed a clinical classification system for MDS (9) which has been generally adopted (Table 1). Several studies have shown that FAB classification system has also a prognostic significance (Table 1) (10). Especially the median survival time is affected by the proportion of blasts in bone marrow.
TABLE 1 ______________________________________ FAB classification of the myelodysplastic syndrome ( 9 ). Median survival is presented as shown by Kouides and Bennett (11). Median survival Subtype Blood Bone marrow (Months) ______________________________________ Refractory anemia (RA) Blasts &lt; 1% Blasts &lt; 5% 32-50 Sideroblasts &lt; 15% Refractory anemia with ring Blasts &lt; 1% Blasts &lt; 5% 45-76 sideroblasts (RARS) Sideroblasts &gt; 15% Chronic myelomonocytic Blasts &lt; 5% Blasts &lt; 20% 11-22 leukaemia (CMML) Monocytes &gt; Promonocytes 1 .times. 10.sup.9 /l Refractory anemia with Blasts &lt; 5% Blasts 5-20% 11-19 excess of blasts (RAEB) Refractory anemia with Blasts &gt; 5% Blasts 21-30% 3-11 excess of blasts in transfor- or or mation (RAEB-t) Auer rods Auer rods ______________________________________
Proposed therapeutic modalities for MDS are extremely divergent including supportive measures, corticosteroids, anabolic steroids, differentiating agents, biologic response modifiers, haematopoietic growth factors, heme arginate, low-dose chemotherapy, intensive chemotherapy, and high dose chemo-radiotherapy with bone marrow transplantation (12).
Efficacy of heme arginate in myelodysplastic syndromes
Heme arginate has been used in treatment of myelodysplastic syndromes by two independent groups of investigators.
Volin et al. (13, 14) gave heme arginate to 26 patients with myelodysplastic syndrome of the subgroups RA, RARS, RAEB, or RAEBt at the dose 2-3 mg hemin/kg in most cases first on four consecutive days and after that once a week for 8 to 12 weeks. Six of the patients showed improvement in cytopenias during the therapy and three of them had long-lasting (&gt;11, &gt;12, and 25 months) good response with normal or close to normal blood cell counts.
Timonen and Kauma (15) treated 14 patients with myelodysplastic syndrome of the subtypes RA, RARS, or RAEB at the dose 3 mg hemin/kg on four consecutive days at two weeks intervals mostly for six treatment cycles. Three of the patients had long-lasting (5, 26, and &gt;41 months) improvement in hemoglobin values and/or platelet counts.
Hemoglobinopathies
Each molecule of hemoglobin consists of four polypeptide (globin) chains and four molecules of heme. There are four different types of the globin chains (.alpha., .beta., .delta., and .gamma.) constituting a hemoglobin molecule in combinations of two plus two. In an adult human the major types of hemoglobin molecules are Hb-A.sub.1 (.alpha..sub.2, .beta..sub.2) and Hb-A.sub.2 (.alpha..sub.2, .beta..sub.2). During the fetal period and during the first year after birth the fetal hemoglobin type Hb-F (.alpha..sub.2, .gamma..sub.2) is also present. Pathological inherited abnormalities in the structure of hemoglobins are called with the general name hemoglobinopathies (16).
More than 100 different abnormal hemoglobins are known, each produced by a mutation affecting one type of polypeptide chain. In sickle cell anemia e.g. the .beta.-globin gene is mutated in a way that one amino acid residue is changed (glu.sub.6 - val.sub.6) in the globin chain. However, this small change affects the function of the hemoglogin and red blood cells to cause a severe disease with hemolytic anemia (16).
The thalassemias are a group of anemias in which the production of .alpha. chain, .beta. chain or .beta. and .gamma. chains is greatly reduced or absent. The severity of thalassemias depend on the subtype and whether the abnormal gene is present with heterozygote or homozygote expression. The .beta.-thalassemia e.g. is often expressed as severe microcytic anemia (16).
An increase in Hb-F in patients with .beta.-thalassemias (e.g. sickle cell anemia, .beta.-thalassemia) may ameliorate the clinical symptoms of the disease. In sickle cell anemia, not only do HbF-containing cells have lower concentration of sickle hemoglobin (Hb-S), but Hb-F directly inhibits polymerization of Hb-S, thus accounting for the lower propensity of such cells to form intracellular polymer and undergo sickling. In .beta.-thalassemia, the elevated .gamma. chain should partially compensate for the deficiency in .beta. chains relative to a chains. Recently, several pharmacological agents, e.g. hydroxyurea and 5-azacytidine, have been used to stimulate Hb-F synthesis in patients with hemoglobinopathies (17, 18, 19, 20).